Extremely sensitive assays are being developed for the detection of pathogenic human retroviruses. These assays will be used for the screening of donated blood and transplantable tissues, in order to prevent the spread of T-lymphotropic leukemia/lymphoma and acquired immunodeficiency syndrome (AIDS). The assays will also be useful for the identification of asymptomatic carriers. The assays employ novel "binary" hybridization probes. If both probes hybridize to their retroviral RNA targets, and only if they hybridize to their targets, a series of enzymatic steps occur that result in the synthesis of replicatable RNA reporters. These reporter molecules are then amplified as much as one-billion-fold by incubation with the RNA-directed RNA polymerase, Q-beta replicase, enabling their easy detection. These assays are well suited for the reliable detection of pathogenic retroviruses in blood and donated tissues. The assays are designed to detect viral nucleic acids, rather than antibodies (which are not present during the early stages of infection). Moreover, the assays are extremely sensitive. The assays take only three hours, so they are particularly compatible with organ transplantation. They are also very simple. All tissue samples, no matter where they come from, are dissolved in a chaotropic salt solution, and are then treated in exactly the same way. No sample fractionation is required. The assays are extremely specific, since two different probes must hybridize to their intended target for signal generation to occur. The assays provide quantitative results over a wide range of target concentrations. They are also readily adaptable to formats designed to simultaneously detect different retroviral species (as well as other transmissible pathogenic agents). And finally, the assays are designed so that they will be compatible with automated devices that handle many different samples simultaneously.